Compositions and methods for treating or preventing oxalate-related disease

ABSTRACT

The present invention comprises methods and compositions for the reduction of oxalate in humans, animals and plants. For example, the invention provides methods and compositions for the delivery of one ore more oxalate-reducing enzymes to the intestinal tracts of persons and animals. The methods and compositions can be used in treating and preventing oxalate-related conditions.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a Continuation of U.S. patent applicationSer. No. 13/919,266, filed Jun. 17, 2013 (now U.S. Pat. No. 10,149,866),which is a continuation of U.S. patent application Ser. No. 10/868,242,filed Jun. 15, 2004 (now U.S. Pat. No. 8,486,389).

FIELD OF THE INVENTION

The present invention relates to compositions and methods for treatingand preventing oxalate related conditions. More particularly, theinvention relates to compositions and methods comprisingoxalate-degrading or oxalate-reducing bacteria and enzymes.

BACKGROUND

Kidney-urinary tract stone disease (urolithiasis) is a major healthproblem throughout the world. Most of the stones associated withurolithiasis are composed of calcium oxalate alone or calcium oxalateplus calcium phosphate. Other disease states have also been associatedwith excess oxalate. These include, vulvodynia, oxalosis associated withend-stage renal disease, cardiac conductance disorders, Crohn's disease,and other enteric disease states.

Oxalic acid, and/or its salt, oxalate, is found in a wide variety offoods, and is therefore, a component of many constituents in human andanimal diets. Increased oxalate absorption may occur after foodscontaining elevated amounts of oxalic acid are eaten. Foods such asspinach and rhubarb are well known to contain high amounts of oxalate,but a multitude of other foods and beverages also contain oxalate.Because oxalate is found in such a wide variety of foods, diets that arelow in oxalate and which are also palatable are hard to formulate. Inaddition, compliance with a low oxalate diet is often problematic.

Endogenous oxalate is also produced metabolically by normal tissueenzymes. Oxalate, which includes dietary oxalate that is absorbed aswell as oxalate that is produced metabolically, is not furthermetabolized by tissue enzymes and must therefore be excreted. Thisexcretion occurs mainly via the kidneys. The concentration of oxalate inkidney fluids is critical, with increased oxalate concentrations causingincreased risk for the formation of calcium oxalate crystals and thusthe subsequent formation of kidney stones.

The risk for formation of kidney stones revolves around a number offactors that are not yet completely understood. Kidney or urinary tractstone disease occurs in as many as 12% of the population in Westerncountries and about 70% of these stones are composed of calcium oxalateor of calcium oxalate plus calcium phosphate. Some individuals (e.g.,patients with intestinal disease such as Crohn's disease, inflammatorybowel disease, or steatorrhea and also patients that have undergonejejunoileal bypass surgery) absorb more of the oxalate in their dietsthan do others. For these individuals, the incidence of oxalateurolithiasis increases markedly. The increased disease incidence is dueto increased levels of oxalate in kidneys and urine, and this, the mostcommon hyperoxaluric syndrome in man, is known as enteric hyperoxaluria.Oxalate is also a problem in patients with end-stage renal disease andthere is recent evidence (Solomons, C. C., M. H. Melmed, S. M. Heitler[1991] “Calcium citrate for vulvar vestibulitis” Journal of ReproductiveMedicine 36:879-882) that elevated urinary oxalate is also involved invulvar vestibulitis (vulvodynia).

Bacteria that degrade oxalate have been isolated from human feces(Allison, M. J., H. M. Cook, D. B. Milne, S. Gallagher, R. V. Clayman[1986] “Oxalate degradation by gastrointestinal bacteria from humans” J.Nutr. 116:455-460). These bacteria were found to be similar tooxalate-reducing bacteria that had been isolated from the intestinalcontents of a number of species of animals (Dawson, K. A., M. J.Allison, P. A. Hartman [1980] “Isolation and some characteristics ofanaerobic oxalate-degrading bacteria the rumen” Appl. Environ.Microbiol. 40:833-839; Allison, M. J., H. M. Cook [1981] “Oxalatedegradation by microbes of the large bowel of herbivores: the effect ofdietary oxalate” Science 212:675-676; Daniel, S. L., P. A. Hartman, M.J. Allison [1987] “Microbial degradation of oxalate in thegastrointestinal tracts of rats” Appl. Environ. Microbiol.53:1793-1797). These bacteria are different from any previouslydescribed organism and have been given both a new species and a newgenus name (Allison, M. J., K. A. Dawson, W. R. Mayberry, J. G. Foss[1985] “Oxalabacter formigenes gen. nov., sp. nov.: oxalate-degradinganaerobes that inhabit the gastrointestinal tract” Arch. Microbiol.141:1-7).

Not all humans carry populations of O. formigenes in their intestinaltracts (Allison, M. J., S. L. Daniel, N. A. Comick [1995]“Oxalate-degrading bacteria” In Khan, S. R. (ed.), Calcium Oxalate inBiological Systems CRC Press; Doane, L. T., M. Liebman, D. R. Caldwell[1989] “Microbial oxalate degradation: effects on oxalate and calciumbalance in humans” Nutrition Research 9:957-964). There are lowconcentrations or a complete lack of oxalate degrading bacteria in thefecal samples of persons who have had jejunoileal bypass surgery(Allison et al. [1986] “Oxalate degradation by gastrointestinal bacteriafrom humans” J. Nutr. 116:455-460). Also, certain humans and animals maymaintain colonies of O. formigenes but nevertheless have excess levelsof oxalate for reasons which are not clearly understood.

What is needed are methods for treating humans and animals to reduce theoxalate levels in their bodies so that oxalate-related conditions aretreated or prevented. Desirable methods would include administration ofoxalate-reducing compositions.

BRIEF SUMMARY OF THE INVENTION

The present invention comprises compositions and methods for treatingand preventing, oxalate-related conditions. Compositions of the presentinvention comprise among others, microorganisms that can reduce oxalateand compositions that comprise enzymes that reduce oxalate. Methods ofthe present invention comprise administering the compositions to treator prevent oxalate-related conditions. One embodiment comprises methodswhich reduce the risk for developing oxalate-related disorders byreducing the amount of oxalate in the intestinal tract. This reductionin the intestinal tract leads to a reduction in systemic oxalate levelsthereby promoting good health.

In one embodiment of the subject invention, a reduction in oxalateabsorption is achieved by supplying oxalate-degrading bacteria to theintestinal tract. In a preferred embodiment, these bacteria areOxalobacter formigenes. These bacteria use oxalate as a growthsubstrate. This utilization reduces the concentration of soluble oxalatein the intestine and, thus, the amount of oxalate available forabsorption. A reduction of oxalate in the intestinal tract can also leadto removal of oxalate from the circulatory system. Methods of thepresent invention contemplate an overall reduction of the oxalate loadin an individual.

In a specific embodiment, the subject invention provides methods andcompositions for the delivery of O. formigenes to the intestinal tractsof persons who are at increased risk for oxalate-related disease. Thesebacteria and their progeny replicate in the intestine and remove oxalatefrom the intestinal tract, thereby reducing the amount of oxalateavailable for absorption and leading to increased oxalate excretion fromthe blood into the intestine.

In accordance with the teaching of the subject invention,oxalate-degrading microbes other than O. formigenes which utilizeoxalate as a substrate can also be used to achieve therapeutic oxalatedegradation thereby reducing the risk of urolithiasis and otheroxalate-related disorders. Such other microbes may be, for example,bacteria such as clostridia or pseudomonads. Additionally, the presentinvention comprises methods and compositions for providing exogenouspolynucleotides capable of conferring oxalate-reducing function tomicroorganisms that can be used to transform nave microorganisms, thoseoriginally unable to reduce oxalate, into microorganisms capable ofreducing oxalate.

In one embodiment of the subject invention, compositions comprise themicrobes that are used to degrade oxalate produce enzymes which conferupon these microbes the ability to degrade oxalate. In an alternativeembodiment, the compositions may comprise microbes that are transformedwith polynucleotide sequences which confer upon the transformed microbesthe ability to degrade oxalate. Polynucleotide sequences that encodeoxalate-reducing genes are contemplated by the present invention.Polynucleotide sequences coding for enzymes found in oxalate-reducingmicroorganisms, such as bacteria or fungi, or other oxalate-reducingenzymes can be used in the methods of the present invention.Polynucleotides may be used to transform cells so that the cells havemore oxalate-reduction activity, the same oxalate-reduction activity, orless oxalate-reduction activity than naturally occurring oxalatereducing microorganisms. Polynucleotides may also be used in syntheticor ex vivo systems to provide proteins having oxalate reducing activity.

The enzymes formyl-CoA transferase and oxalyl-CoA decarboxylase havebeen identified as playing a role in oxalate degradation. Enzymes usedin the methods and compositions of the present invention include, butare not limited to formyl-CoA transferase, oxalyl-CoA decarboxylase,oxalate oxidase, oxalate decarboxylase and other enzymes, cofactors, andco-enzymes that are substituents of oxalate degradation pathways orinvolved in oxalate metabolic pathways, particularly oxalate reduction.

In one embodiment of the subject invention, an appropriate host can betransformed with exogenous polynucleotides encoding these enzyme orenzyme related activities thereby conferring upon the transformed hostthe ability to augment oxalate degradation. The host may be, forexample, a microbe which is particularly well adapted for oraladministration and/or colonizing the intestines. Alternatively, the hostmay be a plant which, once transformed, will produce the desired enzymeactivities thereby making these activities available in the intestinewhen the plant material is consumed. Alternatively, the transformedplant may have a lower amount of oxalate, optionally due to the actionsof the proteins provided by the transformation, and thus when consumed,the plant will not provide as much oxalate to the diet as would anontransformed plant.

The present invention also comprises methods and compositions for plantstransformed with oxalate-degrading or oxalate-reducing enzymes whereinthese plants have enhanced resistance to fungi which require oxalate fortheir pathogenesis of plants or which produce oxalic acid as a mechanismfor their pathogenesis of plants.

The present invention also comprises methods and compositions comprisingenzymes for reducing oxalate levels in order to treat or prevent oxalaterelated conditions. For example, a reduction in oxalate levels isachieved by administering enzymes which act to degrade oxalate. Theseenzymes may be isolated and purified or they may be administered as acell lysate. The cell lysate may be made from any microorganism that hasoxalate-reducing functions, for example, O. formigenes. In a specificembodiment, the enzymes which are administered are one or more of theenzymes of the present invention such as, but not limited to, oxalatedecarboxylase, oxalate oxidase, formyl-CoA transferase and oxalyl-CoAdecarboxylase. Optionally, additional factors which improve enzymeactivity can be administered. These additional factors may be, forexample, oxalyl CoA, MgCl₂, and TPP (thiamine diphosphate, an activeform of vitamin B₁). The compositions comprising enzymes comprise one ormore enzymes, and optionally, cofactors, coenzymes, and other agentsthat enhance enzyme activity, individually or in combination.

In one embodiment of the subject invention, a reduction in oxalatelevels is achieved by administering oxalate-degrading enzymes producedby a recombinant microbe, such as Escherichia coli which has beentransformed to express oxalate-degrading enzymes. The recombinant hostmay be administered in either a viable or non-viable form. A furtheraspect of the subject invention pertains to pharmaceutical compositionsand/or nutritional supplements for oral administration. Thesecompositions release the oxalate degrading microbes, or oxalatedegrading enzymes, in the intestines of humans or animals. Thecompositions of the present invention comprise pharmaceuticallyacceptable formulations. For example, the methods and compositions ofthe present invention comprise a dose delivery system that provides thecompositions to the desired locations, such as delivery of thecompositions to the intestines of the recipient. The compositions of thepresent invention may be administered as a constituent of foods, such asmilk, meats, and yogurt.

In a further embodiment of the subject invention, a reduction in oxalateabsorption is achieved in domesticated, agricultural, or exotic animalsdeficient in oxalate-degrading bacteria by administeringoxalate-degrading microorganisms, plants, and enzymes individually or incombinations

Methods of the present invention comprise treating or preventingoxalate-related conditions in humans and animals by administering aneffective amount of oxalate reducing compositions comprising one or moreoxalate reducing microorganisms, one or more oxalate reducing enzymes orcombination and mixtures thereof. Oxalate-related conditions include,but are not limited to, hyperoxaluria, primary hyperoxaluria, idiopathiccalcium oxalate kidney stone disease (urolithiasis), enterichyperoxaluria, vulvodynia, oxalosis associated with end-stage renaldisease, cardiac conductance disorders, inflammatory bowel disease,Crohn's disease and ulcerative colitis.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A is a graph of data from a high calcium diet.

FIG. 1B is a graph of data from a low calcium diet.

FIG. 2A is a graph of excreted oxalate.

FIG. 2B a graph of excreted oxalate.

FIG. 2C a graph of excreted oxalate.

FIGS. 3A-C a graph of excreted oxalate.

FIG. 4 a graph of excreted oxalate.

DETAILED DISCLOSURE OF THE INVENTION

The present invention comprises methods and compositions for oxalatereduction. The compositions of the present invention comprisemicroorganisms, enzymes, polynucleotide sequences, vectors, cells,plants and animals that are capable of reducing oxalate. Compositionscomprise microorganisms that are capable of reducing oxalate. Suchmicroorganisms include, but are not limited to Oxalobacter formigenes,Pseudomonas, Clostridia, Lactobacilli, Bifidobacteria, some or all ofwhich are capable of reducing oxalate, but also include microorganisms,such as bacteria or fungi that are transformed with exogenouspolynucleotides so that oxalate reducing ability is conferred.Additionally, the microorganisms of the present invention includemicroorganisms that have been transformed with one or moreoxalate-reducing vectors comprising endogeneous or exogeneouspolynucleotide sequences that code for oxalate-reducing enzymes orassociated activities such that the microorganisms are “super reducers”.Super reducers have enhanced native oxalate reducing abilities, forexample, in transformation of Oxalobactor formigenes with additionaloxalate reducing sequences, or microorganisms that do not originallyhave oxalate reducing activity that are transformed with one or moresequences coding for oxalate reducing peptides resulting in enhancedoxalate reducing activity. The oxalate reducing activity encodingsequences may or may not intercalate into the genome or other vectorsfound in the microorganism. Such transformation may include provision ofgene sequences that code for oxalate reducing proteins or peptides ormay provide blocking nucleotides such as antisense or iRNA.

Compositions also comprise enzymes that are components of oxalatereduction pathways. Such compositions comprise one or more enzymes andoptionally include cofactors, coenzymes, and other factors needed ordesired for enzyme activity. Compositions comprise one or more enzymesincluding, but not limited to, oxalate reducing enzymes and otherenzymes involved in oxalate metabolism found in plants, animals orhumans. The compositions comprise one or more of the oxalate reducingenzymes taught herein. As used herein, the term “one or more enzymes”means that one enzyme, such as formyl-CoA transferase is intended, orthat more than one enzyme, for example formyl-CoA transferase andoxalate decarboxylase is intended. As is known in the art, the term doesnot mean one enzyme molecule, but multiples of molecules of one or moreenzyme types.

As used herein, the terms oxalate-degrading enzymes and oxalate-reducingenzymes are interchangeable and both refer to enzymes involved in thereduction or degradation of oxalate in any organism, or to activefragments or recombinant proteins comprising active fragments capable ofreducing or degrading oxalate.

Enzyme compositions of the present invention may also compriseformulations that provide protection of the active enzyme molecules fromdegradation by the stomach or intestinal environment. For example,compositions of enzymes comprise compositions that can protect or cagethe enzymes. The enzymes may be covalently linked to other compounds,including but not limited to PEG. The enzyme may be caged or entrappedwithin a structure such as inside a three dimensional mesh structure,for example, made from polymers that either degrade to release theenzymes or that have pore sizes that allow either the enzyme to leavethe structure or the substrates to penetrate the structure to reach theenzymes. For example, the pore size would allow low molecular weightoxalate and formate to diffuse to the area where the enzymes arepresent. Additionally, the enzymes may or may not be covalently attachedto the polymer structure. Methods and devices for protecting activeenzymes from degradation in proteolytic or other environments harmful toenzymes.

The compositions of the present invention also comprise polynucleotidesequences that encode peptides or proteins that are involved in oxalatereduction pathways. Such polynucleotide sequences can be derived fromany source and can be used in methods known to those skilled in the art,such as for transformation of cells of microbial, plant or animalorigin, and including whole organisms.

Compositions of the present invention also include plants and animalsthat have altered oxalate reduction function. For example, such plantsinclude plants that have been transformed by polynucleotide compositionsso that the amount of oxalate in the plant is lowered or the amount ofoxalic acid produced is increased when compared to untransformed plants.Compositions of the present invention also comprise animals that have anenhanced ability to reduce oxalate. For example, animals having enhancedoxalate reduction abilities can be used as in vivo models for studyingoxalate related conditions.

Methods of the present invention comprise making and using thecompositions of the present invention. Methods of the present inventioncomprise transforming cells, plants and animals by methods known tothose skilled in the art for the introduction of exogenouspolynucleotide sequences. Such polynucleotide sequences can be derivedfrom any source and can be used in methods known to those skilled in theart, such as for transformation of cells of microbial, plant or animalorigin, and including whole organisms. Methods also comprise makingcompositions comprising cell lysates having oxalate reducing activity,compositions comprising one or enzymes having oxalate reducing activity,and compositions comprising dietary constituents made from plants ormicroorganisms having altered oxalate levels.

Methods of the present invention comprise using the compositions of thepresent invention. Such uses include providing polynucleotide sequencesto cells to enhance or repress the oxalate reducing ability of thecells. The present invention comprises methods of administering thecompositions of the present invention to plants or animals for alteringthe oxalate levels of the plant or animal. Methods also include dietarysupplementation methods such that the compositions of the presentinvention are administered to plants or animals in food or fertilizersources or concurrent with food or fertilizer sources to alter theoxalate levels in the food, during the digestion of the food or duringthe uptake by the plants.

Methods of the present invention comprise methods of treating orpreventing oxalate related conditions. Methods comprise administeringthe compositions of the present invention in amounts effective to alterthe oxalate level in an organism. Such methods are effective fortreatment of oxalate conditions in humans and animals including, but notlimited to, hyperoxaluria, primary hyperoxaluria, idiopathic calciumoxalate kidney stone disease (urolithiasis), enteric hyperoxaluria,vulvodynia, oxalosis associated with end-stage renal disease, cardiacconductance disorders, inflammatory bowel disease, Crohn's disease andulcerative colitis.

The subject invention pertains to the introduction of compositionscomprising one or more oxalate-degrading bacteria and/or enzymes into ahuman or animal intestinal tract where the activity of the compositionsreduces the amount and/or concentration of oxalate present therebyreducing the risk of disease due to oxalate.

The present invention comprises methods and compositions for thetreatment and prevention of oxalate-related conditions in humans andanimals. A method for treating oxalate conditions comprisesadministering a composition comprising one or more oxalate-reducingenzymes. Such compositions may be administered one or more times a dayfor one or more days depending on the severity of the oxalate-relatedcondition or the amount of oxalate in the gut or body fluids of thehuman or animal. The treatments may continue as long as unwanted levelsor oxalate are present in the human or animal. For example, the enzymecomposition may be administered one or more times a day for a range oftime including from one day to years. For humans or animals with chronicoxalate-related conditions, the composition may be administered for theentire remaining lifespan of the human or animal.

The methods for treating and preventing oxalate-related conditionscomprise administering a composition comprising an effective amount ofoxalate-reducing enzymes. The amount of enzyme in the compositioncomprises an amount of activity units of oxalate-reducing enzymeactivity that will reduce a portion of the oxalate present in theintestines of the individual or a level of activity units ofoxalate-reducing enzyme activity that will initiate a reduction in theamount of oxalate or maintain a lowered amount of oxalate in theindividual compared to the amount of oxalate present beforeadministration of the composition. The number of activity units ofoxalate-reducing enzyme activity that can be used in a single dosecomposition can range from about 0.0001 units to about 5,000 units, fromabout 5 units to 100 units, and all ranges encompassed therein. Thecompositions may further include other enzymes, cofactors, substrates,coenzymes, minerals and other agents that are helpful in the reductionof oxalate. An unit of the enzyme is the amount of enzyme that willdegrade one micromole of oxalate per minute at 37 C.

In a specific embodiment, the subject invention pertains to methods forthe preparation and administration of compositions comprising cells ofoxalate-degrading bacteria of the species, Oxalobacter formigenes, tothe human or animal intestinal tract where the activity of the microbesreduces the amount of oxalate present in the intestine thereby causing areduction of concentrations of oxalate in the kidneys and in othercellular fluids. In another embodiment, the present invention comprisesmethods for the preparation and administration of compositionscomprising one or more oxalate-degrading enzymes, derived from anysource, to the human or animal intestinal tract where the activity ofthe one or more enzymes reduces the amount of oxalate present in theintestine and lead to a reduction of concentrations of oxalate in thekidneys and in other cellular fluids. The introduced cells or enzymesdegrade oxalate and the bacteria may or may not replicate in theintestinal habitat so that progeny of the initial cells colonize theintestine and continue to remove oxalate. The presence of oxalatereducing bacteria or oxalate reducing bacteria reduces the risk forformation of kidney stones as well as other disease complications causedby oxalic acid. In a preferred embodiment for human use, the specificstrains of O. formigenes used are strains isolated from human intestinalsamples. The strains are thus part of the normal human intestinalbacterial flora. However, since they are not present in all persons, orare present in insufficient numbers, the introduction of these organismscorrects a deficiency that exists in some humans.

Though not wishing to be bound by any particular theory, it is believedthat enrichment of the contents of the intestines with one or morespecies of oxalate-degrading bacteria or oxalate reducing enzymes causesa reduction of oxalate in the intestinal contents. Some of the bacteriaor administered enzymes carry out oxalate degradation at or near thesite of absorption. The activity of the bacteria or administered enzymesdecrease the level of absorption of dietary oxalate. A reduction inoxalate concentration in the intestines can also lead to a removal ofoxalate from cells and the general circulation. More specifically, areduction of oxalate concentration in the intestines can also lead toenhanced secretion of oxalate into the intestine from the blood and thusreduce the amount of oxalate that needs to be excreted in urine. Thus,the methods of the subject invention for administering oxalate reducingbacteria or oxalate reducing enzymes can be used to treat or preventoxalate-related conditions such as primary hyperoxaluria in addition totreatment of dietary hyperoxaluria. The compositions and methods of thesubject invention are particularly advantageous in the promotion ofhealthy oxalate levels in humans and animals.

Pharmaceutical and nutriceutical compositions for the introduction ofoxalate degrading bacteria or one or more oxalate degrading enzymes,alone or in combinations, into the intestine include bacteria or enzymesthat have been lyophilized or frozen in liquid or paste form andencapsulated in a gel capsule or other enteric protection. The gel capmaterial is preferably a polymeric material which forms a delivery pillor capsule that is resistant to degradation by the gastric acidity andenzymes of the stomach but is degraded with concomitant release ofoxalate-degrading compositions by the higher pH and bile acid contentsin the intestine. The released composition then converts oxalate presentin the intestine to harmless products. Pharmaceutical or nutriceuticalcarriers also can be combined with the bacteria or enzymes. These wouldinclude, for example, saline-phosphate buffer. Methods of the presentinvention comprise administration of oxalate-reducing compositions tothe intestinal tract of humans or animals.

Oxalate-reducing compositions comprising one or more oxalate reducingbacteria or one or more oxalate reducing enzymes, or combinations ofbacteria and enzymes, to be administered can be delivered as capsules ormicrocapsules designed to protect the composition from adverse effectsof acid stomach. One or more of several enteric protective coatingmethods can be used. Descriptions of such enteric coatings include theuse of cellulose acetate phthalate (CAP) (Yacobi, A., E. H. Walega,1988, Oral sustained release formulations: Dosing and evaluation,Pergammon Press). Other descriptions of encapsulation technology includeU.S. Pat. No. 5,286,495, which is incorporated herein by reference. Thecompositions of the subject invention can also be formulated assuppositories.

Other methods of administration of these compositions comprising one ormore microorganisms, one or more oxalate reducing enzymes orcombinations and mixtures, to the intestines include adding thecompositions directly to food sources. The one or more bacteria may beadded as freshly harvested cells, freeze dried cells, or otherwiseprotected cells. The one or more enzymes may be added as lyophilizedproteins, encapsulated or microencapsulated enzyme compositions, enzymescomplexed to other materials to maintain activity of the enzymes, andother methods known to those skilled in the art for adding activeenzymes to compositions. Foods may be supplemented with oxalatedegrading compositions without affecting their taste or appearance.These foods may be, for example, yogurt, milk, peanut butter orchocolate. Upon ingestion, when the food products are being digested andabsorbed by the intestines, the oxalate degrading compositions,including one or more microorganisms, one or more enzymes orcombinations, degrade oxalate present in the intestines thus reducingabsorption of oxalate into the bloodstream.

As noted above, a variety of foods can be supplemented with oxalatedegrading compositions. Methods for making such foods containing oxalatereducing compositions include admixing a food material with an oxalatereducing composition. For example, oxalate reducing microbes can begrown in media and separated from the media by, for example,centrifugation. Traditional yogurt cultures obtained from a commercialdairy can be mixed with the oxalate degrading microbial culture. Thismixture of cultures then can be added to the basic dairy yogurt premixwithout adversely affecting taste or consistency. The yogurt can then beproduced and packaged using traditional commercial procedures. Inanother example, the oxalate degrading bacteria can be added to alreadyproduced yogurts. In a similar method, an oxalate reducing compositioncomprising one or more oxalate reducing enzymes can be added to theyogurt bacterial culture or to the yogurt food product.

Another example of the methods of the present invention is to add theoxalate reducing composition to milk after it has been homogenized andsterilized. Such a method is currently used in the dairy industry foradding Lactobacillus acidophilis organisms to milk. Any food sourcecontaining bacteria can be used by supplementing with oxalate-degradingbacteria. These food products include cheese or meat products that havedesirable microorganisms added during processing. Foods comprisingoxalate reducing compositions comprising oxalate reducing enzymes arenot limited to those foods that comprise microorganisms, but include anyfood source in which active enzymes can be added. The materials commonlythought of as food materials can be used as carrier material for theenzymes so that the enzymes are active on oxalate present in the foodmaterial at any stage of production or growth of the food material, orany stage of or digestion by the human or animal, or on oxalate presentin the gut.

In yet a further embodiment, the subject invention provides a novelenzyme delivery system. This system comprises a plant which has beentransformed with heterologous polynucleotide(s) to expressoxalate-degrading enzymes. The enzyme-expressing transgenic plant may beadministered to patients as a constituent of a salad, for example.Further, the enzyme-expressing plant may be administered to animals as aconstituent of feed, for example, or grown in grazing pasture. Theanimals to which these products may be fed include, for example, cattle,pigs, dogs and cats.

Thus, as an alternative method of administration to the intestine,plants are genetically engineered to express oxalate-degrading enzymes.These transgenic plants are added to the diet, with the activity of theenzymes causing a decrease in the presence of oxalate. DNA sequencesencoding these enzymes are known to those skilled in the art and aredescribed in, for example, WO 98/16632.

In addition to plants which can be used as a dietary component topromote healthy oxalate levels in humans or animals, the subjectinvention provides plants with enhanced resistance to microbialinfections. Specifically, the transformed plants of the subjectinvention are protected against microbes which require or use thepresence of oxalate for plant pathogenicity. The plants of the subjectinvention, which are transformed to express oxalate-degrading enzymesare protected against, for example, certain fungi which need oxalate forpathogenicity. The genes encoding the enzymes can be modified to enhanceexpression and/or stability in plants. Also, the expression may be underthe control of promoters which direct expression in particular tissues.

In one embodiment, the strains of bacteria, for example, O. formigenes,used according to the subject invention are pure cultures that areisolated from anaerobic cultures that have been inoculated withdilutions of intestinal contents from normal humans or, for use withanimals, from normal animals. A special calcium oxalate containingmedium that allows detection of oxalate degrading colonies can be used.In one embodiment, the purity of each strain can be assured through theuse of at least two subsequent repetitive cloning steps.

Strains of O. formigenes useful according to the subject invention havebeen characterized based upon several tests, these include: patterns ofcellular fatty acids, patterns of cellular proteins, DNA and RNA(Jensen, N. S., M. J. Allison (1995) “Studies on the diversity amonganaerobic oxalate degrading bacteria now in the species Oxalobacterformigenes” Abstr. to the General Meeting of the Amer. Soc. Microbiol.,1-29), and responses to oligonucleotide probes (Sidhu et al. 1996). Twogroups of these bacteria (Groups I and II, both existing within thepresent description of the species) have been described. Strains usedhave been selected based upon oxalate degrading capacity, and evidenceof the ability to colonize the human intestinal tract. Strains selectedinclude representatives of both Groups I and II of the species.

One embodiment of the present invention involves procedures forselection, preparation and administration of the appropriateoxalate-degrading bacteria to a diversity of subjects. Prominently, butnot exclusively, these are persons or animals which do not harbor thesebacteria in their intestines. These non-colonized or weakly-colonizedpersons or animals are identified using tests that allow for rapid anddefinitive detecting of O. formigenes even when the organisms are atrelatively low concentrations in mixed bacterial populations such as arefound in intestinal contents. The methods of the subject invention canalso be used to treat individuals or animals whose oxalate-degradingbacteria have been depleted due to, for example, antibiotic treatment orin post-operative situations. The methods of the subject invention canalso be used to treat individuals or animals who have colonies ofoxalate-degrading bacteria but who still have unhealthy levels ofoxalate due to, for example, oxalate susceptibility and/or excessiveproduction of endogenous oxalate.

Bacteria which can be used according to the subject invention can beidentified by at least two methods:

1) Oligonucleotide probes specific for these bacteria can be used;and/or

2) A culture test wherein an anaerobic medium with 10 mM oxalate isinoculated and after incubation at 37 .degree. C. for 1 to 7 days, theloss of oxalate is determined.

Pure cultures of O. formigenes strains can be grown in large fermenterbatch cultures and cells can be harvested using techniques known tothose skilled in the art. Cells from a selected single strain ormixtures of known strains can be treated as needed (e.g., freeze driedwith trehalose or glycerol) to preserve viability and are then placed incapsules designed to protect the cells through their passage through theacid stomach (enteric coated capsules).

Cells are ingested in quantities and at intervals determined by theneeds of individuals. In some cases a single, or periodic, use may beall that is needed and in other cases regular ingestion (e.g., withmeals) may be needed.

The invention further pertains to administration to the human or animalintestinal tract of oxalate-degrading products or enzymes prepared fromoxalate reducing organisms such as O. formigenes cells or from othersources, or by methods such as by recombinant means. In one embodiment,oxalate degrading enzymes can be purified and prepared as apharmaceutical or nutriceutical composition for oral consumption. In apreferred embodiment, these enzymes are produced recombinantly. DNAsequences encoding these enzymes are known to those skilled in the artand are described in, for example, WO 98/16632. These sequences, orother sequences encoding oxalate-degrading proteins, can be expressed ina suitable host. The host may be, for example, E. coli or Lactobacillus.The transformed host would include appropriate regulatory andtransporter signals. The expressed protein may be isolated, purified andadministered as described herein. Alternatively, the recombinant hostexpressing the desired oxalate-degrading proteins may be administered.The recombinant host may be administered in either a viable ornon-viable form. In another preferred embodiment, the enzymes are coatedor otherwise formulated or modified to protect the enzymes so that theyare not inactivated in the stomach, and are available to exert theiroxalate-degrading activity in the small intestine. Examples of suchformulations are known to those skilled in the art and are described in,for example, U.S. Pat. No. 5,286,495.

Oxalate degrading enzymes as used herein include all enzymes involved inoxalate pathways and include but are not limited to, oxalate oxidase,oxalate decarboxylase, formyl CoA transferase and oxalyl-CoAdecarboxylase. Oxalate oxidase is expressed in higher plants and itcatalyzes the oxygen dependent oxidation of oxalate to CO₂ withconcomitant formation of H₂O₂. Oxalate oxidases have been purified frommany sources for example, barley seedlings roots and leaves; beet stemsand leaves; wheat germ; sorghum leaves; and banana peel. A rapid threestep purification procedure has been developed to obtain oxalate oxidasefrom barley roots. The gene encoding the barley root oxalate oxidase hasbeen cloned, sequenced and expressed.

Oxalate decarboxylase is mainly present in fungi. A bacterial oxalatedecarboxylase has been recently reported in B. subtilis and is encodedby the yvrk gene. Oxalate decarboxylases catalyze the degradation offree oxalate to CO₂ and formate. This enzyme has been reported inseveral fungi, including Myrothecium, verrucaria, certain strains ofAspergillus niger, and white rot fungus, Coriolus versicolor. The geneencoding the Flammulina velutipes oxalate decarboxylase has been clonedand sequenced; See WO 98/42827.

Oxalyl-CoA decarboxylase is active on a CoA-activated substrate andconverts it into formyl-CoA. A formyl-CoA transferase then acts toexchange formate and oxalate on CoA. These enzymes have been studied inthe oxalate degrading bacteria, Pseudomonas oxalaticus present in thesoil and in Oxalobacter formigenes, residing in the gastrointestinaltract of vertebrates, including humans. O. formigenes has been shown toplay a symbiotic relationship with its host by regulating oxalic acidabsorption in the intestine as well as oxalic acid levels in plasma. Asa result the absence of this bacteria has been found to be a risk factorin oxalate related disorders like recurrent idiopathic calcium oxalateurolithiasis and enteric hyperoxaluria secondary to jejunoileal bypasssurgery, cystic fibrosis and inflammatory bowel disease.

Patents describing various oxalate-degrading enzymes and the genesencoding these enzymes include U.S. Pat. Nos. 5,912,125; 6,090,628; and6,214,980. These patents are incorporated herein by reference in theirentirety as if specifically set forth. The term oxalate-degrading enzymeincludes but is not limited to oxalate oxidase, oxalate decarboxylase,oxalyl-CoA decarboxylase, and formyl-CoA transferase, and includesenzymes that are capable of interacting with oxalate or oxalic acid.These enzymes may be derived from natural sources or synthesized usingrecombinant means known in the art, and include all fragments, such asbinding sites, active sites, or fragments capable of interacting withoxalate or oxalic acid. This term also includes but is not limited toall necessary cofactors, coenzymes, metals, or binding or substratematerials that are needed by the enzyme in interacting with oxalate oroxalic acid. The present invention also contemplates any bindingpartners of these enzymes and includes antibodies and antibody fragmentsthat bind to or interact with the enzymes.

The use of O. formigenes is particularly advantageous because it is ananaerobe that does not grow in aerobic tissue environments and does notproduce any compounds which are toxic to humans or animals. As analternative to either O. formigenes or a recombinant host, otheroxalate-degrading bacteria may be used, such as Clostridium, Bacillussubtilis, Pseudomonas, Lactobacilli, Bifidobacteria. Oxalate-degradingenzymes prepared from such alternative bacteria may be administered orthe entire microbe may be administered.

In addition, all aforementioned embodiments are applicable todomesticated, agricultural, or zoo-maintained animals suffering fromdeficient numbers of oxalate-degrading bacteria, as well as to humans.For example, oxalate-degrading enzymes and/or microbes may beadministered to house pets such as dogs, cats, rabbits, ferrets, guineapigs, hamsters and gerbils, as well as to agricultural animals, such ashorses, sheep, cows and pigs, or wild animals maintained for breedingpurposes such as river otters Many animals that are capable of oxalatereduction lose that ability when captured. The present inventioncomprises methods and compositions for restoring lost or reduced oxalatereducing activity. One aspect of the present invention comprisestreating animals retrieved from the wild that have lost or loweredoxalate reducing activity with the compositions taught herein.

The present invention comprises compositions and methods for theadministration of compositions comprising one or more oxalate-degradingbacteria, one ore more enzymes, or combinations of bacteria and enzymes,into a human or animal gastrointestinal tract. Such compositions andmethods are effective in reducing the amount and/or concentration ofoxalate present. Such methods and compositions are effective in treatingand preventing oxalate related conditions. An aspect of the presentinvention comprises compositions and methods for the introduction ofoxalate-degrading enzymes into the gastrointestinal tract of a human oranimal. The present invention comprises methods for delivering one ormore oxalate-degrading enzymes to the gastrointestinal tract of a humanor animal as pharmaceutical and/or nutriceutical carrier compositions.Such enzymes include, but are not limited to oxalate oxidase, oxalatedecarboxylase, oxalyl-CoA decarboxylase, and formyl-CoA transferase.These enzymes can be derived from sources known to those skilled in theart. For example, the plant enzyme, oxalate oxidase (OXO) can bepurified from Barley seedlings, and oxalate decarboxylase can bepurified from bacterial or fungal sources.

Alternatively the oxalate-degrading enzymes can be derived byrecombinant means. For example, recombinant means such as cloning,expression and purification may be used to obtain oxalate reducingenzymes, for example the B. subtilis oxalate decarboxylase enzyme. Suchrecombinant methods are known to those skilled in the art. For example,disclosed, in general, is the cloning and expression of B. subtilisoxalate decarboxylase (YvrK) gene: The gene for oxalate decarboxylaseprotein (YvrK) has been cloned into the pET-9a and pET-14b plasmid(Novagen, Wis.), under the control of a strong bacteriophage T7promoter, for over-expression as soluble cytosolic protein. Theexpression host was the E. coli strain BL 21(DE3) pLysS, a λDE3 lysogendeficient in proteases and which contains a chromosomal copy of theT7-RNA polymerase gene under the lacUV5 control. In addition, thisstrain carries a pET-compatible plasmid that encodes T7 lysozyme, abifunctional enzyme that cuts a bond in the peptidoglycan layer of thecell wall and inhibits T7 RNA polymerase. This enables greater controlof uninduced basal expression and allows the use of methods that disruptthe inner membrane, such as freeze-thaw, or mild detergents, etc.) toefficiently lyse the cell. Expression of the gene product is induced bythe addition of isopropyl-β-D-thiogalactopyranoside (IPTG). Accordingly,an aspect of the present invention comprises methods comprising theadministration of oxalate-degrading enzymes that have been produced by arecombinant microbe. A variety of expression vectors and hosts can beused to produce oxalate degrading enzymes as recombinant proteins, andsuch methods are known to those skilled in the art.

Another aspect of the present invention comprises methods for reducingoxalate absorption by supplying oxalate-degrading bacteria to thegastrointestinal tract of a human or animal. Such bacteria may include,but are not limited to, Oxalobacter formigenes, Clostridium,Lactobacilli, Bifidobacteria and Pseudomonas. O. formigenes has beenisolated from human fecal specimens and cloned through the selection ofindividual colonies. This includes the isolate HC-1 which was originallyobtained by Ixion Biotechnology in 1996 from Dr. Milton Allison. Forexample, frozen stocks of human strain HC-1, can be used. Methods of thepresent invention comprise enriching of the intestines with one or morespecies of oxalate-degrading bacteria, overall reducing of oxalate inthe intestinal contents, reducing oxalate absorption in the intestines,reducing oxalate concentration in blood and renal fluids and reducingthe deleterious effects on the body due to the presence of oxalate.

Accordingly, an aspect of the present invention comprises compositionsand methods for supplying oxalate-reducing bacteria and oxalatedegrading enzymes, that can reduce oxalate to the intestinal tracts ofpersons having increased risk of oxalate-related diseases and/orconditions. Such diseases and conditions include but are not limited tohyperoxaluria, primary hyperoxaluria, idiopathic calcium oxalate kidneystone disease (urolithiasis), enteric hyperoxaluria, vulvodynia,oxalosis associated with end-stage renal disease, cardiac conductancedisorders, inflammatory bowel disease, Crohn's disease, ulcerativecolitis, persons having undergone jejunoileal bypass surgery, personshaving insufficient concentrations of oxalate-degrading bacteria, andother enteric disease states. Humans and animals that have undergoneantibiotic treatment, chemotherapeutic treatment or other treatmentsthat change the intestinal flora are treated with the compositions andmethods of the present invention. The present invention is used torestore oxalate reduction capability to humans or animals with changedintestinal flora. Increased levels of urinary oxalate excretion promotethe formation of kidney stones, contribute to renal scarring, and mayeven result in kidney failure. Accordingly, an aspect of the presentinvention comprises compositions and methods for reducing the formationof kidney stones.

A reduction in overall oxalate concentrations in the intestines can alsolead to removal of oxalate from cells and general circulation. Morespecifically, a reduction of oxalate concentration in the intestines canalso lead to enhanced secretion of oxalate into the intestine from theblood. Though not wishing to be bound by any particular theory, it iscurrently believed that there is a transepithelial gradient for theenteric elimination of oxalate. Accordingly, an aspect of the presentinvention comprises compositions and methods for lowering blood levelsof oxalate and increasing oxalate excretion by promoting excretion ofoxalate from the blood via a transepithelial gradient of oxalate forcolonic oxalate excretion. A method of the present invention comprisesproviding to the intestines of a human or animal a composition forlowering the oxalate concentration or level of a human or animal. Suchlowering can comprise lowering of the amount of oxalate found in theintestines, in blood, in serum, in tissue fluids, and in other bodilyfluids.

One composition of the present invention comprises an O. formigenespaste prepared for oral administration. For each lot of O. formigenespaste, a single stock vial of HC-1 is used to generate a seed culture inorder to initiate growth in large-scale production fermentation. Thebacteria from each fermentation are collected by centrifugation andblended with cryoprotective excipients, which provide protection againstfreeze-drying. The cell paste can also be subjected to freeze-dryingresulting in a fine powder which has a potency in the range of 10⁷ to10⁹ CFUs/gram. The resulting powder is placed into gelatin capsules thatare enteric coated for safe delivery of the bacteria to the smallintestine.

Compositions of the present invention comprise compositions made fromextracts of one or more oxalate-reducing bacteria in the range fromabout 10³ to about 10¹² cfus/gram, from about 10³ to about 10¹⁰cfus/gram, from about 10⁵ to about 10¹² cfus/gram, from about 10⁵ toabout 10¹⁰ cfus/gram, from about 10⁷ to about 10⁹ cfus/gram, from about10⁷ to about 10⁸ cfus/gram and all ranges in between.

Compositions of the present invention also comprise compositionscomprising one or more enzymes that have activity in reducing oxalate.An aspect of the invention comprises administering an effective amountof an enzyme composition to the gastrointestinal tract of a human oranimal. An effective amount of an enzyme composition is capable ofreducing a portion of oxalate in the intestines or lowering the oxalateconcentration in a human or animal from the level measured prior toadministering the composition. Such measurement may be a measurement ofoxalate present in the gut from food sources or may be a level measuredin a body fluid like blood or urine.

The present invention comprises methods for administering compositionscontaining O. formigenes to the gastrointestinal tracts of a human oranimal. Subjects are preferably dosed with enteric capsules containing≥10⁶ cfus of viable O. formigenes cells. Such dosing preferably occurstwice a day with two major meals. The present invention also comprisesmethods for administering oxalate reducing compositions comprising oneor more oxalate reducing microorganisms, one or more oxalate reducingenzymes or combinations thereof. A method of the present inventioncomprises administering at least one time a day an effective amount ofan oxalate reducing composition wherein the oxalate reducing compositioncomprises one or more oxalate reducing enzymes. Methods also includeadministering such compositions more than one time per day, more thantwo times per day, more than three times per day and in a range from 1to 15 times per day. Such administrations may be continuously, as inevery day for a period of days, weeks, months or years, or may occur atspecific times to treat or prevent oxalate-related conditions. Forexample, a person or animal may be administered oxalate reducingcompositions at least once a day for years to treat or preventoxalate-related conditions or a person or animal may be administeredoxalate reducing compositions at least once a day only at times whenoxalate-containing foods are ingested, or for a restricted time period,such as days or weeks, following procedures or treatments that interferewith normal bacterial flora. Such administration can occur throughroutes known for administration of pharmaceuticals. Administrationthrough oral or intestinal routes, or in combination with food materialsare contemplated by the present invention.

It must be noted that, as used in this specification and the appendedclaims, the singular forms “a”, “an”, and “the” include plural referentsunless the context clearly dictates otherwise.

All patents, patent applications and references included herein arespecifically incorporated by reference in their entireties.

It should be understood, of course, that the forgoing relates only topreferred embodiments of the present invention and that numerousmodifications or alterations may be made therein without departing fromthe spirit and scope of the invention as set forth in this disclosure.

Following are examples which illustrate procedures for practicing theinvention. These examples are not to be construed in any way as imposinglimitations upon the scope of the present invention. On the contrary, itis to be clearly understood that resort may be had to various otherembodiments, modifications, and equivalents thereof which, after readingthe description herein, may suggest themselves to those skilled in theart without departing from the spirit of the present invention and/orthe scope of the appended claims.

EXAMPLE 1

Treatment of High Risk Patients

Primary Hyperoxaluric patients were fed enteric coated capsulescontaining freeze dried powder of O. formigenes twice a day preferablewith their two big meals of the day. Each size-2 capsule contained about137 mg of lyophilized bulk powder containing at least 10⁸ Colony FormingUnits (CFUs)/gram.

For high risk subjects this may be a life long treatment. Subjects inclinical studies showed that colonization dropped when the treatment wasstopped. In the clinical study, treatment was done for 4 weeks and therewas a two week follow up. The 4-week treatment resulted in significantdecrease in blood and urinary oxalate levels as compared to the baselinelevels. But during the follow up period, the stool counts forOxalobacter dropped and the plasma and urine oxalate values started toincrease. Thus, it is proposed that continuous feeding ofoxalate-reducing compositions will be needed to provide the reducedoxalate conditions. Compositions comprising bacteria that can colonizeand establish themselves continuously in the gut could lead to the needfor fewer administrations of oxalate-reducing compositions.

Enteric coated capsules of O. formigenes cells can be ingested bypatient populations at high risk for oxalate related disease. Theseinclude:

1. Persons who produce too much endogenous oxalate due to, for example,a genetic defect like Primary Hyperoxaluria

2. Persons at risk for urolithiasis with high urinary oxalate due toenteric disease (enteric-hyperoxaluria).

3. Persons that have a history of urolithiasis with multiple episodes ofidiopathic stone disease.

4. Persons with high serum oxalate levels due to end stage renaldisease.

5. Persons with vulvar vestibultitis.

6. Persons that have diets with high levels of oxalate such as found incertain areas and seasons in India and in Saudi Arabia. This would alsoinclude individuals who happen to prefer foods such as spinach which arehigh in oxalate.

Anyone of the above described persons or animals are provided acomposition of the present invention. For example, a person with higherthan normal endogenous oxalate levels is treated two times a day, with acapsule designed for delivery of its contents to the large intestine,wherein the capsule contains approximately 10⁶ cfus of O. formigenes.The capsule is preferably given with food.

EXAMPLE 2

Treatment of Low Risk Patients

Enteric protected O. formigenes cells, such as provided in entericcoated capsules can also be ingested by individuals in populations atlower risk for oxalate related disease. It would be desired to colonizethese patients with one or two treatments comprising compositions ofoxalate-reducing materials, such as oxalate-reducing bacteria. Thesepatients could also routinely receive treatments of oxalate-reducingmaterials, either as supplements or as additions to foods such as milkor yogurt. These include:

1. Persons that have lost populations of normal oxalate degradingbacteria due to: treatments with oral antibiotics or bouts of diarrhealdisease.

2. Infants can be inoculated so that a normal protective population ofOxalabacter will be more easily established than is the case later inlife when competitive exclusion principles operate.

The persons or animals who are low risk are treated two times a day,with a capsule designed for delivery of its contents to the largeintestine, wherein the capsule contains at least 10⁷ cfus of one or moreoxalate reducing organisms, such as O. formigenes. The capsule ispreferably given with food.

EXAMPLE 3

Use of Oxalate Degrading Enzymes from Oxalobacter formigenes to ControlHyperoxaluria

A study was conducted to evaluate the efficacy of oxalate degradingenzymes from Oxalobacter formigenes for the control of hyperoxaluria.

Animals Used: Male Sprague Dawley Rats: BW 250-300 g

Diets Used: Normal Diet (N.D.): Harlan Teklad TD 89222; 0.5% Ca, 0.4% P

Drug Used: Lyophilized mixture of Oxalobacter formigenes lysate (sourceof enzymes) with Oxalyl CoA, MgCl₂ and TPP.

Drug Delivery System (Capsules): Size 9 capsules for preclinical ratstudies (Capsu-Gel). Enteric Coating Eudragit L-100-55 (Hulls America,Inc.). Basal 24 hr urine collection. Fecal analysis for Oxalobacterformigenes—rats were not colonized with Oxalobacter formigenes.

Experimental Protocol:

A. Long-term Studies:

Animal Protocol:

Group I (n=4): Fed oxalate diet with lysate. Rats were given twocapsules everyday at 4:00 p.m. and oxalate diet overnight. Diet wasremoved during the day (8:00 a.m. to 4:00 p.m.)

Group II (n=4): Fed oxalate diet as described for Group I (HyperoxaluricControls).

24 hr urine samples were collected on Day 7 and Day 9 of the abovetreatment.

Data on the mean urinary oxalate concentration for the two groups ofrats shown above indicated that feeding of Oxalobacter lysate loweredthe urinary oxalate concentration in Group I rats as compared to thehyperoxaluric controls (Group II). The enzymes can not be active for along duration in the gastrointestinal tract; therefore, short-termstudies were performed as described below.

B. Short-term Studies:

Animal Protocol:

Group I (n=4): Fed 1 capsule at 8:00 a.m.; oxalate diet for two hours(rats were fasted overnight so that they eat well during this period)and 1 capsule at 10:00 a.m.

Group II (n=4): Oxalate diet for two hours as for Group I.

Urine was collected from all the animals for the next five-hour periodand analyzed for oxalate concentration.

This was performed on days 11, 12 and 15 of this study.

The results of this study show that feeding the Oxalobacter lysateproduces a significant decrease in urinary oxalate levels in a 5 hourperiod after oxalate and drug administration in Group I rats as comparedto the hyperoxaluric control group (Group II). At this point a crossoverstudy between the two groups of rats was performed.

C. Cross-Over Studies:

Animal Protocol:

Group I: Fed oxalate diet twice a day at 8:00-10:00 a.m. and 3:00p.m-5:00 p.m.

Group II: Fed 1 capsule twice a day before feeding the oxalate diet asfor Group I.

Short-term studies for the effect of oxalobacter lysate feeding onurinary oxalate levels were performed as described in Section-B above onday-2 and day-5 after the cross-over.

Crossover studies showed that previously hyperoxaluric Group II rats,which were fed the Oxalobacter lysate, showed a decline in urinaryoxalate levels. In contrast the Group-I rats reverted to hyperoxaluriaupon withdrawal of the drug.

EXAMPLE 4

Treatment with Oxalobacter formigenes Cells to Rats

A study was conducted to evaluate the fate of dietary oxalate whenOxalobacter formigenes cells are included in the diet.

Methods:

Male Wistar rats were fed a normal calcium (1%), high oxalate (0.5%)diet, or a low calcium (0.02%), high oxalate diet (0.5%) diet during twoseparate experiments. ¹⁴C-oxalate (2.0 μCi) was given on day 1 and againon day 7 of the study. Oxalobacter formigenes cells (380 mg/d) wereadministered in rat drinking water on days 5-11. The fate of ¹⁴C fromoxalate was measured based on analysis of ¹⁴C in feces, urine andexpired air. The rats served as self controls and measurements duringthe control period (before Oxalobacter cells were fed) were made duringdays 1-4; during the experimental period (when bacterial cells were fed)measurements were made on days 7-11.

Results:

1. When rats were fed the normal (1%) calcium diet, less than 1% of theadministered dose of ¹⁴C from oxalate was recovered in expired air (ascarbon dioxide produced from ¹⁴C oxalate in the intestine, absorbed intoblood and then expired) however in all cases more of the ¹⁴C wasrecovered during the period when rats were fed Oxalobacter cells (FIG.1a ) This is in contrast to results obtained when the diet was low incalcium (0.02%) when more than 50% of the ¹⁴C from oxalate was recoveredas carbon dioxide in expired air during the experimental period whenrats were fed Oxalobacter cells (FIG. 1b ). These results are strikinglydifferent from the very low quantities of ¹⁴C (less than 5%) recoveredduring the control period (before the feeding of Oxalobacter cells).Thus feeding Oxalobacter formigenes cells to rats markedly increased theamount of dietary oxalate that was degraded in the intestinal tract.

2. Feeding Oxalobacter cells also decreased the amount of ¹⁴C-oxalatethat was excreted in urine. Values for a 4 day collections during boththe control and experimental periods and for a single day in each ofthese periods are shown in FIGS. 2a and 2b respectively. Quantities ofoxalate recovered in rat feces were also lower during the experimentalperiod (when Oxalobacter cells were fed) than was found for the controlperiod (FIG. 2c ).

Most laboratory rats do not carry Oxalobacter in their intestinal tracts(they are not colonized). The present results showed that purposefuladministration of these oxalate-degrading bacteria to rats caused alarge portion of the dietary oxalate to be degraded and thatconsequently less of the oxalate from the diet was excreted in urine.

The effects of dietary calcium on oxalate degradation are marked.Calcium complexes with oxalate so that its solubility and availabilityfor attack by Oxalobacter is limited and the amount that is degradedwhen rats are fed a high calcium diet is much less than amounts degradedwhen calcium in the diet is low.

EXAMPLE 5

Effect of Feeding O. formigenes on Urinary Oxalate Excretion in Pigs

Pigs are naturally colonized with Oxalobacter. Decolonization wasachieved in experimental pigs by antibiotic supplementation of the diet.Pigs were fed Oxalobacter in culture broth, which they readily consumed.The pigs were fed a soybean/corn based feed supplemented with 1300 mgoxalate/kg. The basal diet contained 680 mg oxalate/kg. Results areshown in FIGS. 3a-c for three individual pigs.

In all the three pigs urinary oxalate was dramatically decreased duringthe consumption of Oxalabacter. The level of excretion of oxalate inthese pigs decreased to a minimum of approximately 6 mg/g creatinine inall three pigs. This is to be compared with a level of 8-10 mg/gcreatinine that has been observed in humans taking oxalate-free formuladiets. This level is equated to the endogenous synthesis in humans asthe dietary load has been eliminated. It appears that this levelreflects endogenous synthesis in pigs and that the intestinal absorptionhas been eliminated by Oxalobacter treatment. Furthermore, these resultsindicate that the ingested Oxalobacter were able to remove both thecrystalline oxalate added and the food-borne oxalate that wasbioavailable.

In this experiment each pig was fed 1.0 g of cell paste with the morningmeal. At O.D₆₀₀ of 0.6, viable cell count is 2.1.×10⁸ cells/ml, whichextrapolates to 2.1.×10¹³ cells per 100 L. The 100 L fermenter runprovides us on the average 50-60 gm wet wt of cells. Therefore, 1 gm wetwt of cells is about 3.5×10¹¹ viable cells.

The dose of 3.5×10¹¹ viable cells as indicated above could eliminateintestinal absorption of about 2.0 gm of oxalate present per kg diet(1300 mg added oxalate+680 mg present in the diet). The animals consumed1 kg diet per meal.

The body weight of the pigs is about 200 lbs. and the digestive systemof the pigs is believed to be very close to that of humans. In humansthe average daily consumption of oxalate is about 100-400 mg dependingon the diet composition which is also split into three meals/day,therefore on an average a daily dose of 10⁸ to 10¹⁰ viable cells wouldbe sufficient to prevent the dietary absorption of oxalate.

EXAMPLE 6

Effect of O. formigenes Supplementation on Urinary Oxalate Excretion inRats Fed High Oxalate Diet

A study was conducted to determine the effect of the IxOC-3 formulationon colonization status and urinary oxalate levels following a highoxalate diet. An IxOC-3 formulation comprises freeze-dried viable cellsof an oxalate reducing bacteria, such as O. formigenes. The formulationcontains approximately 10⁶-10⁷ cfus/gram per dose. The formulation alsocomprises cyropreservation agents such as trehelose and maltodextrin.

Methods:

Male Harlan Sprague Dawley rats were randomly assigned to 3 groups (6animals/group). Animals of group 1 served as the control group and wereadministered size 9 enteric coated placebo formulation twice daily byoral gavage at a dose level of 10⁰ colony forming units (CFU). Animalsof Groups 2 and 3 were administered Oxalobacter formigenes IxOC-3formulation in size 9 enteric coated capsule form twice daily by oralgavage at dose levels of 10⁶ and 10⁷ CFUs respectively. Capsule gavagewas followed by an autoclaved tap water wash down for all three groups.Following an initial acclimatization period, all groups were feed astandard diet supplemented with 1% oxalate per gram.

Test materials and the placebo control material were prepared followinga standardized protocol. Prior to use, representative samples of eachtest material were analyzed to confirm identity, purity, and potency ofthe test capsules, as well as to confirm the absence of Oxalobacterformagenes in the placebo control material during the dosing period.

Diet was restricted to two daily 1 hour periods starting 15 minutesfollowing morning and evening gavage to ensure capsules were dosed on anempty stomach. Water was provided ad libitum. Food consumption wasrecorded twice daily. Fecal and 24 hour urine samples were collected atDay 1 (prior to oxalate supplemented diet) and weekly thereafter. Theurine data was analyzed via a repeated measures analysis for differencesin mean urinary parameters across dosage groups and time. A dosage groupby time interaction term was also included to assess any possibleinteraction between dosage group and time.

Results:

The results of the analysis indicated there was a statisticallysignificant interaction between dose groups and time (p<0.0001) for allparameters indicating that the urinary parameter profile across time wasdifferent across the dosage groups. To aid in the interpretation of thisinteraction, an analysis of the data was conducted by time point foreach parameter to determine if there was a difference between the dosagegroups with respect to the mean urinary parameters. This analysisrevealed that for the low dose and high dose groups, there was anincrease in urinary oxalate from baseline to 7 days (p<0.0001 bothgroups) but there was no increase from 7 days to 28 days (p=0.1094 lowdose and p=0.6910 high dose). For the placebo group, however, there wasan increase from baseline to 28 days (p=0.0010). Also at day 21 and day28, mean urinary oxalate levels in the placebo Group I weresignificantly higher than those for the low (Group II) and high (GroupIII) dose groups, but no significant difference between the low dose andthe high dose. Thus, there was an overall significant decrease inurinary oxalate excretion in treated rats as compared to rats that werefed the placebo.

EXAMPLE 7

The Effects of Oral Administration of O. formigenes on Urinary OxalateLevels in Patients Suffering from Primary Hyperoxaluria (PH)

Methods:

Nine patients with biopsy proven primary hyperoxaluria (PH) participatedin the study. After receiving initial baseline evaluations, all subjectswere administered Oxalobacter formigenes 1 g cell paste (≥10¹⁰cfus/gram) bid with their main meals for 4 weeks. During this timeperiod, all patients continued to take their normal medication, wereasked to eat their normal diet, and to keep their fluid intake as highas normal. Except for spinach and rhubarb, foods high in oxalate werenot forbidden. Oxolobacter colonization and its influence on urinary andoxalate plasma levels were measured in weeks 5 and 6. Treatment efficacywas followed in terms of urinary oxalate excretion in subjects withnormal renal function and plasma oxalate in subjects with end-stagerenal disease (ESRD).

Results:

1. Treatment demonstrated a significant lowering of urinary oxalate insubjects with normal urine function. Plasma oxalate decreasedsignificantly in seven out of nine subjects. There was a dramaticlowering of plasma oxalate in two subjects with ESRD providing evidencefor enteric elimination of endogenous oxalate into the gut against atrans-epithelial gradient.

2. Consumption of O. formigenes strain HC-1 at dosages ranging from 0.25g to 2.0 g per meal were well tolerated by normal, healthy volunteersreceiving diets containing average or high oxalate levels. A dosage of1.0 gm cell paste twice a day for 28 days was well tolerated by PHpatients.

EXAMPLE 8

Treatment of High Risk Patients with Oxalate Reducing EnzymeCompositions

Primary hyperoxaluric patients are fed one or more enteric coatedcapsules containing a lyophilized oxalate-reducing enzyme composition,comprising oxalate decarboxylase and/or oxalate oxidase, twice a daypreferable with the two main meals of the day. An effective amount ofthe enzyme composition is administered. For example, each size-2 capsulecontains about 5-100 units of each enzyme.

For high risk subjects this is a continuous administration for anextended period of time, probably a life long treatment. Colonizationwill drop when the treatment is stopped.

Enteric coated capsules of oxalate reducing compositions comprisingoxalate reducing enzymes can be administered to patient populations athigh risk for oxalate related disease. These include:

1. Persons who produce too much endogenous oxalate due to, for example,a genetic defect like Primary Hyperoxaluria

2. Persons at risk for urolithiasis with high urinary oxalate due toenteric disease (enteric-hyperoxaluria).

3. Persons that have a history of urolithiasis with multiple episodes ofidiopathic stone disease.

4. Persons with high serum oxalate levels due to end stage renaldisease.

5. Persons with vulvar vestibultitis.

6. Persons that have diets with high levels of oxalate such as found incertain areas and seasons in India and in Saudi Arabia. This would alsoinclude individuals who happen to prefer foods such as spinach which arehigh in oxalate.

Anyone of the above described persons or animals are provided acomposition of the present invention. For example, a person with higherthan normal endogenous oxalate levels is treated two times a day, with acapsule designed for delivery of its contents to the large intestine,wherein the capsule contains approximately an equivalent effectiveamount of an enzyme composition having enzyme activity similar to thatprovided by 10⁷ cfus of an oxalate-reducing bacterium, such as O.formigenes. The capsule is preferably given with food.

EXAMPLE 9

Treatment of Low Risk Patients with Oxalate Reducing Enzyme Compositions

Enteric protected oxalate reducing compositions comprising a mixture ofthe oxalate reducing enzymes oxalate decarboxylase and/or oxalateoxidase, such as provided in enteric coated capsules can also beadministered to individuals in populations at lower risk foroxalate-related disease or at risk for oxalate-related conditions. Aneffective amount of the enzyme composition is administered in thedesired treatment regimen.

It would be desired to administer the compositions to these patientseither for shorter periods of time when they are at risk foroxalate-related conditions or simultaneously with materials thatcontribute to oxalate-related condition. These patients could alsoroutinely receive treatments of oxalate-reducing compositions, either assupplements or as additions to foods such as milk or yogurt. Theseinclude persons that have lost populations of normal oxalate degradingbacteria due to: treatments with oral antibiotics or bouts of diarrhealdisease, or infants.

The persons or animals who are low risk are treated two times a day,with a capsule designed for delivery of its contents to the largeintestine, wherein the capsule contains an effective amount of theenzyme composition. For example, each size-2 capsule contains about5-100 units of each enzyme. The capsule is preferably given with food.

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and the scope of the appended claims.

What is claimed is:
 1. A method for reducing systemic circulatingoxalate in a subject in need thereof, comprising orally administering acomposition comprising oxalate decarboxylase in an amount effective toreduce the amount or concentration of oxalate in the gastrointestinaltract of said subject, wherein the administering of said composition iseffected at least once per day for a period of months or years, whereinthe method is effective to reduce systemic circulating oxalate in saidsubject.
 2. The method of claim 1, wherein the subject is a humansubject.
 3. The method of claim 1, wherein the oxalate decarboxylase isproduced recombinantly.
 4. The method of claim 1, wherein thecomposition further comprises one or more components selected fromcoenzymes, cofactors, substrates, and other substituents of the oxalatedegradation pathways.
 5. The method of claim 1, wherein the oxalate isfrom dietary sources.
 6. The method of claim 1, wherein the oxalate isfrom endogenous sources.
 7. The method of claim 1, wherein the subjectis suffering from an oxalate-related condition.
 8. The method of claim7, wherein the oxalate-related condition is hyperoxaluria.
 9. The methodof claim 7, wherein the oxalate-related condition is selected fromprimary hyperoxaluria, enteric hyperoxaluria, urolithiasis, vulvodynia,and oxalosis associated with end-stage renal disease.
 10. The method ofclaim 9, wherein the oxalate-related condition is primary hyperoxaluria.11. The method of claim 9, wherein the oxalate-related condition isenteric hyperoxaluria.
 12. The method of claim 7, wherein theoxalate-related condition is selected from, cardiac conductancedisorders, inflammatory bowel disease, Crohn's disease, and ulcerativecolitis.
 13. The method of claim 1, wherein the subject has undergonegastrointestinal surgery.
 14. The method of claim 1, wherein the subjecthas undergone antibiotic treatment.
 15. The method of claim 1, whereinthe subject has undergone bariatric surgery.